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Chromosome analysis in hematologic disorders
Chromosome Analysis in Hematologic Disorders The Leukemias
AVERY A. SANDBERG, M.D. RODMAN MORGAN, M.S. CAROL BERGER, M.T. (A.S.C.P.) BARBARA KAISER-McCAW HECHT, Ph.D. FREDERICK HECHT, M.D. Tempe, Arizona
From the Genetics Center and Cancer Center of Southwest Biomedical Research Institute, Temps. Arizona. Dr. Sandberg is a Visiting PhysicianScientist at the Southwest Biomedical Research Institute from Roswell Park Memorial Institute, Buffalo, New York. Requests for reprints should be addressed to Dr. Avery A. Sandberg, Department of Genetics and Endocrinology, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, New York 14263. Manuscript accepted November 22, 1983.
.For two decades, cytogeneticstudieshave been used to rule in (or out) the Philadelphia (Phi) chromosomeassociated with chronic myeloidleukemia. Beyondthis stngtepurpose,&mmo%x~ studies have generally not been utilized in or applied to the practice of hematology-oncology.This report presentsmale and female patients, teens to 70s in age, with representative hematologic disorders,in whom the cytogenetic findingswere useful clinically. These cases illustratethe followingprinciples:(1) hematologicdisorderscan be characterized by chromosomeanalysis; (2) chromosomeftndings help in the diagnosis,prognosis,and treatment of blood diseases; (3) bloodand bone marrowsamplescan be processedroutinelyfor cytogenetic analysis; (4) these samples can be transported long distances from clinic to laboratory; and (5) the contemporary practice of hematologyand oncolagyrequireschromosomeanalysts for fuller evaluation and understandingof hematologic conditions. Hundreds of research reports have appeared since 1960 on chromosomes in hematologic disorders [ 11. A growing number of specific chromosome changes have been found to characterize hematologic entities. These chromosome markers have been discovered to be pertinent to the diagnostic, prognostic, therapeutic, and survival aspects of blood diseases [ l-31. The utilization of new chromosome knowledge in hematology and oncology, however, has received scant attention. The aim of this report is to illustrate the practical usefulness of chromosome analysis and show how the interpretation of q&genetic (chromosomal, karyotypic) information is helpful to hematology and oncology in practice. In this report, we wish to present some of our experience in servicing cytogenetically not only the area of metropolitan Phoenix but also other areas of Arizona, as well as clinics outside the state, with particular attention being given to illustrative cases in which the collaboration between the cytogenetic laboratory and clinician led to a more precise definition of clinical cases and a clearer understanding of the patients’ diseases. Also, this report has been written with the hope that similar arrangements can be made in other clinical centers in which the clinicians have neither access to high-level cytogenetic laboratories in their own institutions nor the type of information and help that clinicians should be seeking and receiving from cytogeneticists. Only through such approaches will it be possible to utilize the cytogenetic findings to more fully understand the hematologic disorders.
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CHROMOSOME ANALYSIS IN HEMATOLOGIC DISORDERS-SANDBERG
TABLE I
ET AL
Clinical and Cytogenetic Data on Seven Patients with tiematoiogic Disorders
Patient Ageand Number Sex 1
60F
2
71F
3
55M
4
61M
5
19M
6 7
66M 77M
Diagnosis Chronic myelocytic leukemia in blastic phase Chronic myelocytic leukemia Acute nonlymphocytic leukemia Malignant lymphoma Acute myelomonocytic leukemia
Pancytopenia Myeloproliferative disorder
Numberof Cells Examined
Abnormal Cells (percent)
14
100
46,XX,t(9*;22~q32;ql2),t(16;16)(q24;q23)/46,XX,t(Q*:22), t( 16; 16),t(X;lO)(q26;q24)‘inv(Q)
15
100
29
56
46,XX,t(9;22)(q32;ql2),del(9~q22),del(13~q12)/46,XX,t(9; 22),Qq46,XY,t(6;21)(q22;q22),del(Q)(q22)
14
57
13
100
14 19
64 91
PATiENTS AND METHODS Obtaining Specimens. Blood samples were obtained (5 to 10 cc) from patients using (1) a sterile heparinized (green-top) vacuum tube or (2) a heparinized syringe with prompt transfer of blood into a sterile plain (red-top) vacuum tube. Bone marrow was aspirated directly into a syringe that had been previously rinsed with heparin. The initial aiiquot of 0.5 to 1.O cc of bone marrow was transferred promptly into (1) a sterile screw-top vial containing 2 cc of sterile tissue culture medium or (2) a sterile, small (2 to 3 cc), plain (red-top) vacuum tube. Transport of Specimens to the Laboratory. The samples of blood and/or bone marrow were not refrigerated, since we have generally found that samples, especially of bone marrow, provide better cytogenetic results when not refrigerated. Within the environs of Phoenix, samples were brought to our laboratory usually on the same day they were obtained. We used a commercial delivery service instructed to protect the samples from the extremes of blistering sun in summer and the cool of the desert in winter. Delivery to the laboratory occurred usually within two hours. Shipping from longer distances required reliance on express transportation of one type or another. Among the services we have found satisfactory are Federal Express and Purolator. The United States Postal Service has not proved reliable in the expeditious delivery of samples. Shipment was initiated one day and delivery took place at the laboratory by the following day. Generally, the personnel involved in such shipments were cognizant of untoward effects on the samples of delayed delivery. Samples so shipped consistently arrived within 24 hours at the laboratory. Laboratory Methods. The chromosome constitutionof blood and bone marrow cells was determined by established iaboratory methods [ 1,4]. We will not describe these methods here in detail, since we would like to place special emphasis
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Karyotype
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45,XX,-22,lp+,dup(lqKqPl-q23),14q+,i(3q),t(6p;22q), (p11.2;q11.2) 46,XY,t(2;5Xpl3;q35)‘46,XY,t(2;5)/47,XY,+7,t(2;5),t(15; 16)/76,Xx,-Y,t(2;5),+1,+2,+3,+4,+5,+5,+6,+7, +7,+6,+6,+9,+9,+10,+10,+11,+12, +12,+13,+14,+15,+15,+16,+17,+16,+19,+19, +20,+21,+21,+22,+22 45,XY,-l2,t(7p;l2q),16p+,lQp+ 46,XY,lp+/47,XY,lp+.+lp+
on obtaining and shipping blood and bone marrow samples, which are the responsibilities of the hematologist/oncologist, i.e., of the clinician. Chromosome analysis of marrow and blood samples was performed following 24 to 48 hours of incubation at 37’C without phytohemaggiutinin. G banding was utilized for detailed karyotypic analysis. Patients. We have selected seven patients for presentation to illustrate points that may be useful. The seven patients were chosen from among more than 500 we have studied. The experience reported herein is neither unique nor different from that of other laboratories involved in chromosome analysis of hematologic disorders. CASE REPORTS, CYTOGENETIC COMMENTS
FINDINGS,
AND
In Table I are shown the cytogenetic data and some of the clinical findings in the seven patients studied. Patient 1. A 80-year-old woman had known chronic myelocytic leukemia diagnosed in October 1977, with a white blood cell count of 38,OOO/pi, a platelet count of 317,00O/pl, and a hemoglobin level of 14.4 g/dl. The leukocyte differential revealed 55 percent segmented cells, 10 percent band forms, 22 percent metamyelocytes, 3 percent myeiocytes, and 8 percent lymphocytes. When the patient was first examined cytogenetically, the leukemic ceils contained a Ph’ transiocation (9;22) involving a chromosome 9, which had a (q)(pl lq13) inversion. Subsequently, the patient had rather unusual cytogenetic findings that were compatible with development of the blastic phase (Figure 1). These findings, seen in a sample obtained on June 14, 1982, consisted of a mixture of ceils in the blood, i.e., those with 18;18 transiocation (57 percent) and others with (18;18)(q24;q23) and (X; iO)(q28;q27) translocations (43 percent). The blood cells were terminal deoxyribonucleotffl transferase-negative. The appearance of the two additional translocations heralded the development of an acute leukemia-like phase, a finding that
CHROMOSOME
ANALYSIS
IN HEMATOLOGIC
DISORDERS-- SANDBERG ET AL
Figure 1. Karyotype (G banded) of a leukemic blood cell from Patient 1 with chronic myelocytic leukemia. The Ph’ chromosome is due to 9;22 translocation (broken arrows), although chromosome 9 is abnormal by having an inversion (see Figure 2). Translocations 16; 18 (thin arrows) and X; 10 (thick arrows) were present and pointed to the blastic phase of chronic myelocytic leukemia.
is not unusual in the blastic phase of chronic myelocytic leukemia, i.e., the appearance of new karyotypic changes not observed during the chronic phase [I]. Gl 1 staining of a sample obtained on September 9, 1982 confirmed that the chromosome 9 involved in the Ph’ translocation (Figure 2) had an inversion of the centromeric heterochromatin, this probably being a constitutive phenomenon. Upon receiving the cytogenetic report indicative of the blastic phase, the patient’s physician prescribed hydroxyurea, vincristine, and prednisone therapy for three weeks, which resutted in aplasia of the marrow. Two weeks after treatment was discontinued, the white blood cell count was 10 to 12 X 103 with a differential compatible with the chronic phase of chronic myelocytic leukemia. One week later, the patient began to receive daunorubicin and cyclophosphamide therapy for a week, at the end of which the marrow was again
aplastic. Therapy was stopped. The patient died in October 1982, at which time the chronic myelocytic leukemia was in the blastic phase. Comment: Ph’-positive chronic myelocytic leukemia is a disease that is usually associated with a relatively long chronic phase and a short blastic phase. The latter is generally resistant to therapy and, hence, is the most serious phase of the disease. When any new chromosomal changes (in our patient, the two translocations) in addition to the Ph’ appear, they almost invariably herald or are associated with the development of the blastic phase [ 1,5]. The most common of these changes are an extra Phi, trisomy 8, and a 17q isochromosome. Our patient is of special interest since the karyotypic changes were of unusual nature. The Ph’ translocation involved a chromosome 9 with an inversion. The Ph’ translocation did not involve the chromosome 9 without
,
igure 2. G- 11 banding of a leukemic cell of Patient 7 showing (arrows) the inverted segment of one chromosome 9 (upper one).
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CHROMOSOME ANALYSIS IN HEMATOLOGIC DISORDERS-SANDBERG
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Figure 3. Karyotype (G banded) of a leukemic cell from Patient 2 with Phipositive chronic myelocytic leukemia. Two anomalies, 9q- (thick arrow) and 13q- (thin arrow), appeared when the disease became complicated by thrombocythemia but not by an overt blastic phase of myeloid or lymphoid nature. The Ph’ chromosome has resulted from the usual translocation between chromosomes 9 and 22 (broken arrows).
the inversion in any of the cells examined, indicative that the chronic myelocytic leukemia was of clonal origin, i.e., emanating from a cell with an inversion of chromosome 9, which through an active process of proliferation replaced almost all the cells in the marrow. The translocations, 16; 18 and X; 10, observed in the cells of our patient are rather unique and have not been hitherto described in chronic myelocytic leukemia. In fact, involvement of the X chromosome in translocations other than the Ph’ in chronic myelocytic leukemia is very rare (described in only three patients to date), and that of chromosomes 10,16, and 18 has been observed in only one patient, three patients, and one patient, respectively [6]. It is possible that the relatively favorable response to chemotherapy observed in our patient may be related to the establishment of the blastic phase on the basis of the chromosome changes and initiation of early therapy. Of course, when more specific and successful therapy for the blastic phase becomes available, the use of the cytogenetic findings to ascertain impending or developing blastic phase may be crucial in the success of such therapy. Exceptions to what has just been stated do occur, as exemplified by Patient 2 to be described next, in whom the appearance of additional chromosomal changes was not clearly associated with a blastic phase. It is possible that, in these patients, the chromosomal changes are not detrimental to the cellular biology, and the blastic phase does not supervene. Patient
2.
myelocytic
originally
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This patient, a 71-year-old woman with chronic is of interest, for when leukemia was diagnosed, she was shown to have Phi-positive leukemia,
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chronic myelocytic leukemia with the usual (9;22)(q32;q12) translocation. Over the years, a 9q--,(9)(q22) deletion, anomaly developed in addition to the Phi, and more recently a 13q-, (13)(q12) deletion, developed as well, in almost 100 percent of the cells (Figure 3). The 9q- anomaly was first seen in the marrow on April 30, 1981 and the 13q- in a marrow sample obtained on January 28, 1982. The patient’s disease was diagnosed on June 17,1976, at which time her white blood cell count was 30,7OO/pl and the platelet count 719,100/@; she was treated with busulfan from 1976 to 1980. In September 1980, a definite increase in the platelet number was observed (1.7 millionl~l) and she began to receive melphalan, which she had been taking up to the time of cytogenetic examination in early 1981. However, no evidence of a blastic phase has been observed; thus, in this patient, the 9q- anomaly appears not to have been associated with an overt blastic phase, although it is possible that the chromosomal change may have preceded the clinical changes by a significant period of time. Attempts at treating the thrombocythemia, which appeared in early 1982, with hydroxyurea, uracil mustard, plasmapheresis, iron dextran, and actinomycin D resulted in the appearance of sepsis, probably due to the development of concomitant severe leukopenia (white blood cell count less than 1,OOO/pl), necessitating cessation of the therapy as no effect on the platelet count was observed. On June 30, 1982, and, again on August 3, 1982, injection of 32P resulted in a reduced platelet count (8 X 105/pl) and a stable white blood cell count (3,500/~1), with improvement in the patient’s condition. However, at the time of this writing thrombocythemia has again developed (platelet count 1.7 to 2.6 X 106/~I). No evidence of the blastic phase has appeared.
Comment: This case of Ph’-positive chronic myelocytic leukemia has several interesting facets, from both a cytogenetic and clinical viewpoint. The appearance of two additional chromosomal changes,
CHROMOSOME
ANALYSIS
IN HEMATOLOGIC
DISORDERS--SANDBERG
ET AL
b
Figure 4. Karyotype (G banded) of a leukemic cell from Patient 3 with acute nonlymphocytic leukemia. The major karyotypic change is an 8;2 1 translocation (broken arrows) seen in the M-2 type of acute myeloblastic leukemia; the 9qanomaly (solid arrow) usually suggests a graver prognosis when the anomaly appears, as it did in this patient.
i 1 i j 1 / 1
14
”
15
lb
r
rJ * m
20
-
i.e., 9q- and 13q-, was not associated with the development of the blastic phase. However, concomitant with the development of the chromosome changes, thrombocythemia, which was not present during the initial four years of her disease, became a distressing part of the chronic myelocytic leukemia and has been difficult to control. It is possible that the thrombocythemia represents an unusual manifestation of a blastic phase, constituting in a way an increased proliferation of megakaryocytic elements, instead of the usual increase in myeloblasts and related cells seen in a bona fide blastic phase. Patient 3. This patient, a 52%year-oldman with acute nonlymphocytic leukemia, is of interest because his case points to the value of obtaining chromosome analysis in the follow-up of such patients. The patient’s history consisted of fatigue, weakness, and easy bruising for a period of about a month prior to examination. On January 15, 198 1, his white blood cell count was 45,4OO/~l with 29 percent blasts (containing Auer rods) in the blood, hemoglobin level 7.9 g/dl, hematocrit 25 percent, and platelet count 67,OOOl~l. The patient began to receive chemotherapy (daunomycin, cytosine arabinoside, and 6thioguanine); complete remission was achieved as evidenced by the normal blood picture on March 10, 1981, with a white blood cell count of 9,8OO/pl and no blast forms, a hemoglobin level of 22.3 g/dl, hematocrit of 34 percent, and platelet count of 2OO,OOO/@l.However, on April 20, 1981, when the patient was admitted for consolidation chemotherapy, the white blood cell count was 5,600/& the hematocrit 36 percent, and the platelet count 285,OOO/pl; the bone marrow contained 23 percent of cells with the abnormal karyotype. In July, the patient had an asymptomatic relapse of the disease. During the summer of 1981, the patient received 12 platelet transfusions because of severe thrombocytopenia,
-
but the platelet count never rose above 25 X lo”/pl. The white blood cell count during that period ranged from 1,5OO/pl to 6,0OO/~l. The patient died on August 27, 1981. When originally seen (January 16, 1981), the patient had a mixture of cells in the blood and bone marrow consisting of cytogenetically normal cells, as well as those having the (8;21)(q22;q22) translocation, not an uncommon finding in acute myeloblastic leukemia of the M-2 variety, but also being accompanied by deletion of the long arm of chromosome 9 (9q-) at band q22 (Figure 4). Generally, patients with the 8;21 translocation have a better prognosis than most other patients with acute nonlymphocytic leukemia [ 1,2], have more favorable responses to therapy and, hence, show and have maintenance of complete remission more readily than patients with other forms of acute nonlymphocytic leukemia. However, in the presence of other karyotypic changes, in this case 9q-, the outlook for these patients changes radically, for they tend to have rather short remissions and survival. In fact, this was the case in our patient, who began to receive chemotherapy in midJanuary 1981, had complete remission about mid-March of that year, but had a partial relapse a month later and an overt relapse in July 198 1. Cytogenetic analysis performed on marrow samples before July (on April 20, 1981 and May 28, 1981) indicated relapse of the leukemia by the appearance of a substantial number of cells with the chromosomal changes just described, i.e., 8;21 translocation and 9q- deletion; thus, the bone marrow contained 23 percent blast forms in April, 50 percent in May, and nearly 100 percent in July. On July 24, 1981, the marrow showed exclusively cells with the 8;21 translocation and 9q- deletion.
Comment: The 8;21 translocation has been shown to characterize a specific subgroup of patients with acute nonlymphocytic leukemia, i.e., acute myeloblastic leukemia of the M-2 type. These patients with acute myeloblastic leukemia have a relatively good prognosis
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CHROMOSOME
ANALYSIS
IN HEMATOLOGIC
DISORDERS-SANDSERG
ET AL
Flgure 5. Karyotype (G banded) of a leukemic blood cell from Patient 4 with malignant lymphoma. included in the abnormalities are a 74qi- (thick arrow}, an i(3q) (thln arrow), and a translocation between chromosomes 8 and 22 (curved arrow).
when compared with other patients with acute nonlymphocytic leukemia, readily have responses to therapy, and have long complete remissions, and usually have Auer bodies in the leukemic cells. However, when additional karyotypic changes appear, the clinical course usually worsens and the disease becomes difficult to control with chemotherapy. In Patient 3, the appearance of the 9q- anomaly was associated with progression of the disease. Patient 4. A 61-year-old Hispanic male gardener was admitted with a possible diagnosis of acute myeloblastic leukemia or related disorder, made on the basis of bone marrow examination. He was admitted to the Palo Alto Veterans Administration Hospital on June 12, 1982 from another hospital for an evaluation of “hypersplenism and chronic lymphocytic leukemia.” The patient had a history of malaise, lethargy, intermittent epistaxis, fever and chills, diaphoresis, and cough for three to four months, accompanied by a weight loss (12 to 15 pounds) due to decreased appetite over the preceding six months. When first seen, the patient had “massive splenomegaly and an abnormal complete blood count.” On admission to the Palo Alto Veterans Administration Hospital, the patient had no hepatomegaly but did have splenomegaly with the spleen edge at the iliac crest. Before admission, the white blood cell count was 14,500/@ with 11 percent blasts, 14 percent promyelocytes, 35 percent lymphocytes, 14 percent atypical cells, 32 percent segmented cells, 2 percent monocytes, 6 percent eosinophils, and IO percent “smudge” cells; the hemoglobin level was 9.0 g/dl, and the hematocrit was 27.6 percent. Bone marrow biopsy and aspiration specimens obtained on June 14, 1982 contained “multiple histiocytes among many abnormal-looking lymphocytes with indented and elongated nuclei.” The blood cells were terminal deoxyri-
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bonucleotidyl transferase-negative. The diagnosis of Lennett’s lymphoma was agreed upon. Treatment with cyclophosphamide, vincristine, procarbazine, and prednisone led to a greatly improved clinical state, although at the time the patient decided to go home, he still had a white blood cell count of 23,1OO/pl with 12 percent polymorphonuclear cells, 1 percent band forms, 78 percent lymphocytes, 3 percent monocytes, 2 percent eosinophils, and 4 percent promyelocytes, a hematocrit of 30 percent, and a platelet count of 84,OOOI~l. Karyotyping of blood cells examined on June 15, 1982 after incubation without phytohemagglutinin revealed the following: 46,XY/45,XY,-22,lp+,dup(lq)(q21+q23), i(3q),t(8;22)(pl1.2;q11.2),14q+ (Figure 5). The source of the extra material on the long arm of chromosome 14 could not be established with certainty. The results of the cytogenetic studies were obtained at a time when the exact diagnosis in this patient was uncertain. The results indicated that in all probability the patient did not have acute or another type of leukemia but probably some type of lymphoma, as based on the presence of the 14q+ anomaly, which is extremely rare in acute myeloblastic leukemia and other types of acute nonlymphocytic leukemia [ 71. Subsequent re-examination of the abnormal cells in the blood and a re-evaluation of the nature of the cells in the marrow revealed the most likely diagnosis to be malignant lymphoma, mixed cell type with high content of epithelial cells. Thus, examination of the bone marrow revealed it to be of normal cellularity. The myeloid-to-erythroid ratio was low [ 1,8]. The differentiation of the granulocytes and erythroblasts was normal. Sixty-two percent of the nucleated cells seemed to be abnormal lymphocytes (10 to 12 p in diameter) with basophilic cytoplasm, fine reticular chromatin, indented nuclei, and several nucleoli. These abnormal cells appeared to be compatible with those seen in lymphosarcoma cell leukemia or lymphoma. The disease has responded well to
CHROMOSOME
Figure 6. Blood and marrow leukemic cells of Patient 5. The monocytoid nature of the cells is evidenced by the folded nuclei and general appearance of the cells (Wri@t’s stain; original magnifi~tion X 1,200, reduced by 35 percent).
ANALYSIS
IN HEMATOLOGIC
DISORDERS--SANDBERG
ET AL
-
therapy and is in complete remission at the time of this writing. Comment: The 14q-t anomaly has been shown to be the most common and consistent karyotypic change in various lymphomas, the incidence being particularly high in lymphomas of B cell origin, although 14q+ has also been seen in T cell lymphomas [ 1,7,8]. The origin of the extra material on chromosome 14 varies and the donor chromosome may ultimately be shown to have specificity, akin to that of chromosome 8 in Burkitt’s lymphoma and chromosome 18 in follicular lymphoma. The translocation between chromosomes 8 and 22 is rare in acute nonlymphocytic leukemia and is more likely to occur in lymphoma. In our patient, the short arm of chromosome 8 was involved in the translocation, whereas in most lymphomas with involvement of this chromosome, the long arm is characterized by a break at band q24. The other cytogenetic changes observed in our patient (lp+,duplq,i3q) may occur in both myeloid and lymphoproliferative disorders [l] and, thus, cannot be used as differential points in the diagnosis. In toto, the chromosomal changes observed in this patient are rather characteristic of lymphoma, in this case a lymphoma of mixed cell type. Patient 5. This patient, a 19-year-old man, has presented a rather interesting clinical condition, in that when he was admitted, the exact diagnosis was not readily ascertained. The patient was first seen on April 19, 1982, with a history of anorexia and left flank pain of very short duration. At that time, the blood findings showed a white blood cell count of 118,OOO/yl. The patient denied any other symptoms such as night sweats, fever, bone pain, or unusual bleeding. Past history was noncontributory.
On physical examination, the patient was a well-developed young man with a few ecchymoses. The liver was definitely enlarged (8 cm below the costal margin) and the tip of the spleen was palpable with a splenic rub being audible. Some adenopathy was also found. At the time of examination, the patient’s hemoglobin level was 14.9 g/dl, the hematocrit 41 percent, and the white blood cell count 157,3OO/~l with approximately 80 percent atypical cells. These cells were pleomorphic, with some appearing of lymphoid nature and others more myelomonocytic. The platelet count was 155,OOO/~l. During the patient’s hospitalization, the white blood cell count rose to nearly half a million in a week; bone marrow examination revealed relatively good erythropoiesis and presence of megakaryocytes. However, a large number of abnormal cells was present and these are thought to be myelomonoblastic in nature. Chemical stains of the peripheral blood suggested monocytic disease, as only testing for nonspecific esterase activity showed positive results. Periodic acid-Schiff, Sudan black, peroxidase, and specific esterase stains all demonstrated negative findings. Terminal deoxyribonucleotidyl transferase activity was very low. During the ensuing days, the spleen definitely increased in size, the hematocrit dropped to 34 percent, and the platelet count remained in the range of 150,000/~1. Treatment with daunomycin, cytosine arabinoside, and thioguanine was initiated, and within five days, his white blood cell count dropped to 22,9OO/pl. A reevaluation of the hematologic smears of the blood and marrow revealed about 70 percent of the cells in the blood to be mononuclear with slightly blue cytoplasm (Figure 6). The cells resembled to some extent those seen in S&ary syndrome or other forms of leukemic lymphoma or lymphosarcoma. A bone marrow specimen obtained on April 19, 1982 was shown to be markedly hypercellular with 30 percent of the nucleated cells belonging to the abnormal series described before. About 30 percent of the cells belonged to
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ET AL
Figure 7. Karyotype (G banded) of a marrow cell from Patient 5 with acute myelomonocytic leukemia is shown at /eft, and the original chromosomal abnormality present in the leukemic cells of this patient is shown at right. The abnormality consisted of a (2;5)(p23;q35) translocation. At left are shown the karyotypic changes seen during a recent relapse (December 3, 7982}, i.e., the original 2;5 translocation (thin arrows) and a new translocation involving chromosomes 15 and 16 (thick arrows). In addition, partial trisomy of a chromosome 7 (left) consisting of duplication of 923-33 and + 7 are also present.
the erythroblastic series. The megakaryocytes appeared to be somewhat increased. The patient is now thought to definitely have had acute myelomonocytic leukemia with recent central nervous system involvement requiring the intrathecal administration of cytosine arabinoside. The latter led to a much improved status, with the maintenance therapy consisting of cytosine, vincristine, and prednisone. Throughout this period, the bone marrow has shown a remission picture. The patient had a relapse in early December 1982, including impressive lymphadenopathy and skin lesions. Since then, his condition has been in and out of remission, with each relapse being characterized by the aforementioned skin lesions and adenopathy. Considerable pleomorphism of the cells was observed in the blood during relapse, large cells being present in substantial number. Cytogenetic examination of the unstimulated (no phytohemagglutinin) blood cells in a sample obtained on April 20, 1982 revealed an abnormal karyotype consisting of a translocation between chromosomes 2 and 5, i.e., (2;5) (p23;q35) translocation (Figure 7). This finding appeared to be more compatible with those translocations observed in some forms of acute nonlymphocytic leukemia than in lymphoma. In particular, involvement of chromosome 5 occurs with a high frequency in acute nonlymphocytic leukemia [9, lo]. Furthermore, the translocation of the type observed in this patient, i.e., 2;5 translocation, and the lack of other chromosomal changes, combined to indicate that the most likely diagnosis in this patient was an acute leukemia of soma form, probably of the monocytic variety. Blood cells stimulated with phytohemagglutinin had a normal male karyotype.
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At the beginning of December 1982, the karyotypic findings in this patient progressed and underwent a radical change. The pseudodiploid cells seen previously, although still present, constituted a minority of the metaphases, with cells containing 47 (33 percent) and 78 (30 percent) chromosomes predominating. The cells with 47 chromosomes, in addition to the 2;5 translocation, developed a (15;16)(q22;p13.3) translocation (Figure 8) whereas the cells with 78 chromosomes, although still containing the 2;5 translocation, did not have the 15;16 translocation and showed only numeric changes consisting of extra chromosomes in every autosomal group, an extra X and a missing Y. The hyperploid cells with 78 chromosomes contained four normal number 15 and three normal number 16 chromosomes. Thus, it appears that in this patient, two divergent karyotypic progressions occurred, one with the 15; 16 translocation and the other through numeric changes only; however, the leukemic origin of the cells is evidenced by the presence of the 2;5 translocation, seen in the originally examined material, in both new clones. It is possible that extramedullary relapse of the acute myelomonocytic leukemia occurred in this patient (in lymph nodes, skin) and that the hyper-triploid clone with 78 chromosomes originated thusly and accounted for the pleomorphic blood cell picture. Comment: Acute myelomonocytic leukemia is a somewhat complicated entity in that this group of leukemias may be rather heterogeneous in character; thus, it is not surprising that the cytogenetic findings have not revealed any specificity akin to that in acute myeloblastic leukemia, acute monoblastic leukemia, and
CHROMOSOME ANALYSIS IN HEMATOLOGIC DISORDERS-SANDBERG
ET AL
1
&
7
a
6s Figure 8. Katyotype (G banded) of a leukemic cell of Patient 6, containing a number of complicated chromosomal changes (see Figure 9). Arrows point to the three abnormal chromosomes whose genesis is shown schematically in Figure 9.
4: ..
.Q”
19
20
I
-------.~
(p14;ql l),t(7;16)(p14;pl3.3) (Figure 8). Thus, the short arm of chromosome 12 was lost and most of the long arm of chromosome 12 was translocated to an abbreviated chromosome 7. Part of the short arm of chromosome 7 was translocated to the short arm of chromosome 16 and another part to the short arm of chromosome 19 (Figure 9). The
acute promyelocytic leukemia [ 1,2,11]. In some patients, involvement of the long arm of chromosome 11 (1 lq-) has been described and in these the monocytoid element is rather clear and evident [ 121. Monosomy of chromosome 7 is frequent in acute myelomonocytic leukemia [ 1,9, lo] and may be indicative that such cases are possibly secondary in nature, since -7 has been shown to be a reflection of a leukemia secondary to drugs or toxic agents [9, lo]. The chromosomal findings in our patient, i.e., 2;5 translocation, are of interest, for they have not been described in acute myelomonocytic leukemia, although involvement of chromosome 5 is not rare in acute nonlymphocytic leukemia, particularly in leukemias complicating other diseases. Both 5qand -5 anomalies are seen often in secondary leukemias due to either radiation or chemotherapy [ 9, lo].
complexity of the chrornosotnal changes is rather unique and pointed to a definite leukemic process in operation. Comment:
The involvement
of chromosomes
5, 7,
Pallent 6. A 66-year-old man, a retired railroad boxcar cleaner (probably exposed to carbon tetrachloride), was first seen for severe pancytopenia and thought to have a preleukemic or leukemic condition. At the time of diagnosis, the bone marrow was found to be hypoplastic for erythroid, myeloid, and megakaryocytic elements, with mature lymphocytes, prolymphocytes, and a few lymphoblasts being seen in the marrow. The platelet count at that time was 45,OOO/~l, the white blood cell count 1,OOo/l.~lwith IO percent segmented cells, the hemoglobin level 5.5 g/dl, and the hematocrit 18 percent. The chromosomal findings determined on September 14, 198 1 were complex and quite interesting. The marrow contained a mixture of normal and abnormal cells, with the most likely description of the abnormal karyotype being 45,XY,-l2,del(l2)(qter~qll),t(7;19)(p21;p13.5),t(7;12)
12
1
16
19
7gure 9. Schematic presentation of the karyotypic changes seen in the leukemic cells of Patient 6 shown in Figure 8. As shown, the short arm of one chromosome 12 was lost, with the long arm being translocated to an abbreviated short arm of a chromosome 7. The latter had lost most of its short arm through translocations to chromosomes 16 and 19, with the part to chromosome 16 being inverted.
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CHROMOSOME
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Figure 10. Karyotype (G banded) of a marrow cell of Patient 7 with a myeloproliferative disorder (? preleukemia). The abnormalities consist of two abnormal (1p-l) number 1 chromosomes (arrows).
and 12 in Patients 5 and 6 may be indicative that the hematologic processes in these two cases, i.e., acute leukemias, may be related to some toxic exposure, since the involvement of these chromosomes has been shown to be rather frequent in these conditions [%lOl. The most likely interpretation of the chromosomal findings in Patient 6 is that they resulted from a toxic agent (perhaps carbon tetrachloride), which led to the bone marrow hypoplasia and what is probably a background of a smoldering secondary leukemia. Patient 7. A 77-year-old man presented with a complicated clinical picture of either preleukemia or an unusual myeloproliferative disorder, the latter shown subsequently to be myelofibrosis. Bone marrow biopsy performed on June 25, 1982 revealed alternating areas of hypercellularity and increased fibrosis. Treatment with allopurinol was begun. The karyotype was shown to be 46,XY/46,XY,lp+ (6 percent of
the cells) and 46,XY,lp+,lp+ (85 percent of the cells) (Figure 10). These karyotypic findings pointed to a definitely malignant hematologic disease, probably in the myeloid series, primarily based on the frequent involvement of chromosome 1 in such patients.
At the time of the chromosome analysis, the patient’s white blood cell count was 16.6 X 103, the marrow very fibrotic, and the clinical picture thought not to be that of a preleukemia. The patient has been uncooperative both regarding any further diagnostic procedures or regarding follow-up. As of the date of this writing, the patient’s condition has remained pretty much the same, with a hemoglobin level of 10 g/dl, a white blood cell count of 17,OOO/~l without blasts in the blood, and moderate hepatosplenomegaly.
Comment: Involvement of chromosome 1 is one of the most frequent in hematologic disorders [ 1,131, including trisomy (i- 1) of the chromosome and morpho-
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logic changes of either the short or long arm, but much more often of the long arm. Our Patient 7 is of further interest because some of the cells contained an extra copy of the 1p-t chromosome. Thus, in this patient, the chromosome changes pointed to a malignant hematologic disorder in the general category of a dysmyelopoietic or myeloproliferative syndrome, but its exact nature remains uncertain. COMMENTS This study demonstrated several “practical” points, among which is the feasibility of sending blood and marrow samples for cytogenetic analysis from considerable distances (e.g., Oakland and Palo Alto, California). The success rate in obtaining optimal cytogenetic information with such samples has been high. A crucial factor is to determine the express system of transportation that works expeditiously and reliably for a particular location; it is important to “educate” the personnel involved in transporting and delivering the samples about such crucial facets as the necessity for speed, the need to avoid freezing the samples (or overheating them), and the aim of having the samples in the cytogenetic laboratory within 24 hours or less. With proper attention and interest on the part of all involved in obtaining and shipping of the blood or marrow samples, this system can and has been made to work in Phoenix and Buffalo. There is no reason why a similar system cannot be established in other centers in the United States or other countries. Other points raised and demonstrated in this report are those associated with each of the seven representative cases of hematologic disorders in which the
CHROMOSOME ANALYSIS IN HEMATOLOGICDISORDERS-SANDBERG ET AL
in bone marrow and/or blood samples of patients who live considerable distances from a cytogenetic laboratory. Certainly, the information gained is more than worth the cost and effort. Even though establishment of the presence or absence of a Ph’ chromosome in chronic myelocytic leukemia has become a “routine” procedure in hematology and, in fact, failure to perform this analysis may constitute a form of malpractice in some locations, clinicians have not taken advantage of a similar approach in other hematologic disorders, particularly the acute leukemias. The practical application of karyotypic findings in the diagnostic and prognostic aspects of the acute leukemias has been spelled out in several international workshops on the chromosome changes in these diseases [2,3,5]. Yet, only a minority of oncologists and hematologists have taken advantage of the cytogenetic information in the acute leukemias and related disorders. There are a number of reasons for this situation, as compared with the determination of the Ph’ chromosome in chronic myelocytic leukemia, which possibly account for the infrequency of performing chromosome analysis in acute leukemia and similar disorders. These include the more technically difficult side of examining the chromosomes in acute leukemia cells versus those of chronic myelocytic leukemia, relatively complex karyotypic changes in acute leukemia, as well as their more complex interpretation, and lack of appreciation of the meaning and nature of the cytogenetic abnormalities in the acute leukemias. Hopefully, the results presented herein will convince clinicians to avail themselves of what we think is a critical and crucial aspect of leukemia management.
cytogenetic findings were useful diagnostically and therapeutically. Briefly, the following patients were presented: Patient 1: chronic myelocytic leukemia with a Phi chromosome and two additional translocations, 16; 18 and X;lO, heralding an imminent blastic phase; Patient 2: chronic myelocytic leukemia with a Ph’ chromosome and two marker chromosomes (9q- and 13q-) associated not with the blastic phase but with thromobocythemia; Patient 3: acute myeloid leukemia of the M-2 type with an 8;21 translocation and then a 9q- marker indicating imminent relapse; Patient 4: malignancy of uncertain type with a 14q+ marker chromosome indicative, not of acute myeloblastic leukemia, but of lymphoma; Patient 5: leukemia of uncertain type with a 2;5 translocation consistent with acute nonlymphocytic leukemia, specifically acute myelomonocytic leukemia; Patient 6: pancytopenia probably due to exposure to organic solvents, with 12q- marker and three translocations (7; 12, 7; 16, and 7; 19) indicative of smoldering secondary leukemia; and Patient 7: myelofibrosis, with a lpi- marker associated with a dysmyelopoietic disease. In each of the seven cases presented, the exact diagnosis was not readily apparent when the patients were first seen or when a change in the clinical condition occurred. In several patients, confusion due to the complicated histologic features, cinical condition, or signs prevented the pathologist and oncologist from arriving at a specific diagnosis with confidence. Examination of the chromosomes was of considerable help, i.e., ruling out a diagnosis being considered in several cases, pointing to the type of disease present in others, and establishing the diagnosis with specificity in some. Thus, the utilization of cytogenetic data, when considered in conjunction with the clinical, pathologic, and laboratory data regarding a particular patient, can be of considerable value in the management of patients with various blood disorders. It is hoped that cytogenetic studies of patients with hematologic disorders will find an ever-widening application, particularly, as already indicated, since such studies can be readily performed
ACKNOWLEDGMENT This study would not have been possible without the collaboration of the following clinicians: Teresita C. Barnett, Sun City, Arizona; Robert D. Ligorsky, Phoenix, Arizona; Jerry A. Olshan, Phoenix, Arizona; Donald E. Rediker, Palo Alto, California; Philip P. Scheerer, Phoenix, Arizona.
REFERENCES 1. 2.
3.
4.
Sandberg AA: The chromosomes in human cancer and leukemia. New York: Elsevier North-Holland, 1980. Second International Workshop on Chromosomes in Leukemia (1979): Cytogenetic, morphologic, and clinical correlations in acute non-lymphocytic leukemia with t(8q--:21q+). Cancer Genet Cytogenet 1980; 2: 99-102. Third International Workshop on Chromosomes in Leukemia (1980): Chromosomal abnormalities in acute lymphoblastic leukemia: structural and numerical changes in 234 cases. Cancer Genet Cytogenet 1981; 4: 101-110. Sandberg AA, Abe S: Cytogenetic techniques in hematology. Clin Haematol 1980; 9: 19-38.
5.
6.
7.
8.
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First International Workshop on Chromosomes in Leukemia, Helsinki, Finland (1977): Cancer Res 1978; 38: 867868. Sandberg AA: The X chromosome in human neoplasia. ineluding sex chromatin and congenital conditions with X chromosome anomalies. In: Sandberg AA, ed. The cytogenetics of the mammalian X chromosome. New York: Alan R. Liss, 1983; 459-498. Mitelman F, Levan G: Clustering of aberrations to specific chromosomes in human neoplasms. IV. A survey of 1,871 cases. Hereditas 1981; 95: 79-139. Yunis JJ, Oken MM, Kaplan ME, Ensrud KM, Howe RR,
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Theologides A: Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkin’s lymphoma. N Engl J Med 1982; 307: 1231-1236. Rowley JD, Golomb HM, Vardiman JW: Nonrandom chromosome abnormatiii in acute l&emia and dysmyelopoietic syndromes in patients with previously treated malignant disease. Blood 1981; 58: 759-767. Sandberg AA, Abe S, Kowalczyk JR, Zedgenidze A, Takeuchi J, Kakati S: Chromosomes and causation of human cancer and leukemia. L. Cytogenetics of leukemias complicating
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11.
12.
13.
other diseases. Cancer Genet Cytogenet 1982; 7: 95136. Hagemeijer A, H&hlen K, Sizoo W, Abels J: Transkxation (9; 1 l)(p21;q23) in three cases of acute monoblastic leukemia. Cancer Genet Cytogenet 1982; 5: 95-105. Berger R, Bemheim A, Weh HJ, Daniel MT, Flandrin G: Cytogenetic studies on acute monocytic leukemia. Leuk Res 1980; 4: 119-127. Rowley JD: Abnormalities of chromosome no. 1 in haematological malignancies. Lancet 1978; I: 554-555.
AVERY A. SANDBERG, M.D. RODMAN MORGAN, M.S. CAROL BERGER, M.T. (A.S.C.P.) BARBARA KAISER-McCAW HECHT, Ph.D. FREDERICK HECHT, M.D. Tempe, Arizona
From the Genetics Center and Cancer Center of Southwest Biomedical Research Institute, Temps. Arizona. Dr. Sandberg is a Visiting PhysicianScientist at the Southwest Biomedical Research Institute from Roswell Park Memorial Institute, Buffalo, New York. Requests for reprints should be addressed to Dr. Avery A. Sandberg, Department of Genetics and Endocrinology, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, New York 14263. Manuscript accepted November 22, 1983.
.For two decades, cytogeneticstudieshave been used to rule in (or out) the Philadelphia (Phi) chromosomeassociated with chronic myeloidleukemia. Beyondthis stngtepurpose,&mmo%x~ studies have generally not been utilized in or applied to the practice of hematology-oncology.This report presentsmale and female patients, teens to 70s in age, with representative hematologic disorders,in whom the cytogenetic findingswere useful clinically. These cases illustratethe followingprinciples:(1) hematologicdisorderscan be characterized by chromosomeanalysis; (2) chromosomeftndings help in the diagnosis,prognosis,and treatment of blood diseases; (3) bloodand bone marrowsamplescan be processedroutinelyfor cytogenetic analysis; (4) these samples can be transported long distances from clinic to laboratory; and (5) the contemporary practice of hematologyand oncolagyrequireschromosomeanalysts for fuller evaluation and understandingof hematologic conditions. Hundreds of research reports have appeared since 1960 on chromosomes in hematologic disorders [ 11. A growing number of specific chromosome changes have been found to characterize hematologic entities. These chromosome markers have been discovered to be pertinent to the diagnostic, prognostic, therapeutic, and survival aspects of blood diseases [ l-31. The utilization of new chromosome knowledge in hematology and oncology, however, has received scant attention. The aim of this report is to illustrate the practical usefulness of chromosome analysis and show how the interpretation of q&genetic (chromosomal, karyotypic) information is helpful to hematology and oncology in practice. In this report, we wish to present some of our experience in servicing cytogenetically not only the area of metropolitan Phoenix but also other areas of Arizona, as well as clinics outside the state, with particular attention being given to illustrative cases in which the collaboration between the cytogenetic laboratory and clinician led to a more precise definition of clinical cases and a clearer understanding of the patients’ diseases. Also, this report has been written with the hope that similar arrangements can be made in other clinical centers in which the clinicians have neither access to high-level cytogenetic laboratories in their own institutions nor the type of information and help that clinicians should be seeking and receiving from cytogeneticists. Only through such approaches will it be possible to utilize the cytogenetic findings to more fully understand the hematologic disorders.
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CHROMOSOME ANALYSIS IN HEMATOLOGIC DISORDERS-SANDBERG
TABLE I
ET AL
Clinical and Cytogenetic Data on Seven Patients with tiematoiogic Disorders
Patient Ageand Number Sex 1
60F
2
71F
3
55M
4
61M
5
19M
6 7
66M 77M
Diagnosis Chronic myelocytic leukemia in blastic phase Chronic myelocytic leukemia Acute nonlymphocytic leukemia Malignant lymphoma Acute myelomonocytic leukemia
Pancytopenia Myeloproliferative disorder
Numberof Cells Examined
Abnormal Cells (percent)
14
100
46,XX,t(9*;22~q32;ql2),t(16;16)(q24;q23)/46,XX,t(Q*:22), t( 16; 16),t(X;lO)(q26;q24)‘inv(Q)
15
100
29
56
46,XX,t(9;22)(q32;ql2),del(9~q22),del(13~q12)/46,XX,t(9; 22),Qq46,XY,t(6;21)(q22;q22),del(Q)(q22)
14
57
13
100
14 19
64 91
PATiENTS AND METHODS Obtaining Specimens. Blood samples were obtained (5 to 10 cc) from patients using (1) a sterile heparinized (green-top) vacuum tube or (2) a heparinized syringe with prompt transfer of blood into a sterile plain (red-top) vacuum tube. Bone marrow was aspirated directly into a syringe that had been previously rinsed with heparin. The initial aiiquot of 0.5 to 1.O cc of bone marrow was transferred promptly into (1) a sterile screw-top vial containing 2 cc of sterile tissue culture medium or (2) a sterile, small (2 to 3 cc), plain (red-top) vacuum tube. Transport of Specimens to the Laboratory. The samples of blood and/or bone marrow were not refrigerated, since we have generally found that samples, especially of bone marrow, provide better cytogenetic results when not refrigerated. Within the environs of Phoenix, samples were brought to our laboratory usually on the same day they were obtained. We used a commercial delivery service instructed to protect the samples from the extremes of blistering sun in summer and the cool of the desert in winter. Delivery to the laboratory occurred usually within two hours. Shipping from longer distances required reliance on express transportation of one type or another. Among the services we have found satisfactory are Federal Express and Purolator. The United States Postal Service has not proved reliable in the expeditious delivery of samples. Shipment was initiated one day and delivery took place at the laboratory by the following day. Generally, the personnel involved in such shipments were cognizant of untoward effects on the samples of delayed delivery. Samples so shipped consistently arrived within 24 hours at the laboratory. Laboratory Methods. The chromosome constitutionof blood and bone marrow cells was determined by established iaboratory methods [ 1,4]. We will not describe these methods here in detail, since we would like to place special emphasis
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45,XX,-22,lp+,dup(lqKqPl-q23),14q+,i(3q),t(6p;22q), (p11.2;q11.2) 46,XY,t(2;5Xpl3;q35)‘46,XY,t(2;5)/47,XY,+7,t(2;5),t(15; 16)/76,Xx,-Y,t(2;5),+1,+2,+3,+4,+5,+5,+6,+7, +7,+6,+6,+9,+9,+10,+10,+11,+12, +12,+13,+14,+15,+15,+16,+17,+16,+19,+19, +20,+21,+21,+22,+22 45,XY,-l2,t(7p;l2q),16p+,lQp+ 46,XY,lp+/47,XY,lp+.+lp+
on obtaining and shipping blood and bone marrow samples, which are the responsibilities of the hematologist/oncologist, i.e., of the clinician. Chromosome analysis of marrow and blood samples was performed following 24 to 48 hours of incubation at 37’C without phytohemaggiutinin. G banding was utilized for detailed karyotypic analysis. Patients. We have selected seven patients for presentation to illustrate points that may be useful. The seven patients were chosen from among more than 500 we have studied. The experience reported herein is neither unique nor different from that of other laboratories involved in chromosome analysis of hematologic disorders. CASE REPORTS, CYTOGENETIC COMMENTS
FINDINGS,
AND
In Table I are shown the cytogenetic data and some of the clinical findings in the seven patients studied. Patient 1. A 80-year-old woman had known chronic myelocytic leukemia diagnosed in October 1977, with a white blood cell count of 38,OOO/pi, a platelet count of 317,00O/pl, and a hemoglobin level of 14.4 g/dl. The leukocyte differential revealed 55 percent segmented cells, 10 percent band forms, 22 percent metamyelocytes, 3 percent myeiocytes, and 8 percent lymphocytes. When the patient was first examined cytogenetically, the leukemic ceils contained a Ph’ transiocation (9;22) involving a chromosome 9, which had a (q)(pl lq13) inversion. Subsequently, the patient had rather unusual cytogenetic findings that were compatible with development of the blastic phase (Figure 1). These findings, seen in a sample obtained on June 14, 1982, consisted of a mixture of ceils in the blood, i.e., those with 18;18 transiocation (57 percent) and others with (18;18)(q24;q23) and (X; iO)(q28;q27) translocations (43 percent). The blood cells were terminal deoxyribonucleotffl transferase-negative. The appearance of the two additional translocations heralded the development of an acute leukemia-like phase, a finding that
CHROMOSOME
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Figure 1. Karyotype (G banded) of a leukemic blood cell from Patient 1 with chronic myelocytic leukemia. The Ph’ chromosome is due to 9;22 translocation (broken arrows), although chromosome 9 is abnormal by having an inversion (see Figure 2). Translocations 16; 18 (thin arrows) and X; 10 (thick arrows) were present and pointed to the blastic phase of chronic myelocytic leukemia.
is not unusual in the blastic phase of chronic myelocytic leukemia, i.e., the appearance of new karyotypic changes not observed during the chronic phase [I]. Gl 1 staining of a sample obtained on September 9, 1982 confirmed that the chromosome 9 involved in the Ph’ translocation (Figure 2) had an inversion of the centromeric heterochromatin, this probably being a constitutive phenomenon. Upon receiving the cytogenetic report indicative of the blastic phase, the patient’s physician prescribed hydroxyurea, vincristine, and prednisone therapy for three weeks, which resutted in aplasia of the marrow. Two weeks after treatment was discontinued, the white blood cell count was 10 to 12 X 103 with a differential compatible with the chronic phase of chronic myelocytic leukemia. One week later, the patient began to receive daunorubicin and cyclophosphamide therapy for a week, at the end of which the marrow was again
aplastic. Therapy was stopped. The patient died in October 1982, at which time the chronic myelocytic leukemia was in the blastic phase. Comment: Ph’-positive chronic myelocytic leukemia is a disease that is usually associated with a relatively long chronic phase and a short blastic phase. The latter is generally resistant to therapy and, hence, is the most serious phase of the disease. When any new chromosomal changes (in our patient, the two translocations) in addition to the Ph’ appear, they almost invariably herald or are associated with the development of the blastic phase [ 1,5]. The most common of these changes are an extra Phi, trisomy 8, and a 17q isochromosome. Our patient is of special interest since the karyotypic changes were of unusual nature. The Ph’ translocation involved a chromosome 9 with an inversion. The Ph’ translocation did not involve the chromosome 9 without
,
igure 2. G- 11 banding of a leukemic cell of Patient 7 showing (arrows) the inverted segment of one chromosome 9 (upper one).
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Figure 3. Karyotype (G banded) of a leukemic cell from Patient 2 with Phipositive chronic myelocytic leukemia. Two anomalies, 9q- (thick arrow) and 13q- (thin arrow), appeared when the disease became complicated by thrombocythemia but not by an overt blastic phase of myeloid or lymphoid nature. The Ph’ chromosome has resulted from the usual translocation between chromosomes 9 and 22 (broken arrows).
the inversion in any of the cells examined, indicative that the chronic myelocytic leukemia was of clonal origin, i.e., emanating from a cell with an inversion of chromosome 9, which through an active process of proliferation replaced almost all the cells in the marrow. The translocations, 16; 18 and X; 10, observed in the cells of our patient are rather unique and have not been hitherto described in chronic myelocytic leukemia. In fact, involvement of the X chromosome in translocations other than the Ph’ in chronic myelocytic leukemia is very rare (described in only three patients to date), and that of chromosomes 10,16, and 18 has been observed in only one patient, three patients, and one patient, respectively [6]. It is possible that the relatively favorable response to chemotherapy observed in our patient may be related to the establishment of the blastic phase on the basis of the chromosome changes and initiation of early therapy. Of course, when more specific and successful therapy for the blastic phase becomes available, the use of the cytogenetic findings to ascertain impending or developing blastic phase may be crucial in the success of such therapy. Exceptions to what has just been stated do occur, as exemplified by Patient 2 to be described next, in whom the appearance of additional chromosomal changes was not clearly associated with a blastic phase. It is possible that, in these patients, the chromosomal changes are not detrimental to the cellular biology, and the blastic phase does not supervene. Patient
2.
myelocytic
originally
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This patient, a 71-year-old woman with chronic is of interest, for when leukemia was diagnosed, she was shown to have Phi-positive leukemia,
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chronic myelocytic leukemia with the usual (9;22)(q32;q12) translocation. Over the years, a 9q--,(9)(q22) deletion, anomaly developed in addition to the Phi, and more recently a 13q-, (13)(q12) deletion, developed as well, in almost 100 percent of the cells (Figure 3). The 9q- anomaly was first seen in the marrow on April 30, 1981 and the 13q- in a marrow sample obtained on January 28, 1982. The patient’s disease was diagnosed on June 17,1976, at which time her white blood cell count was 30,7OO/pl and the platelet count 719,100/@; she was treated with busulfan from 1976 to 1980. In September 1980, a definite increase in the platelet number was observed (1.7 millionl~l) and she began to receive melphalan, which she had been taking up to the time of cytogenetic examination in early 1981. However, no evidence of a blastic phase has been observed; thus, in this patient, the 9q- anomaly appears not to have been associated with an overt blastic phase, although it is possible that the chromosomal change may have preceded the clinical changes by a significant period of time. Attempts at treating the thrombocythemia, which appeared in early 1982, with hydroxyurea, uracil mustard, plasmapheresis, iron dextran, and actinomycin D resulted in the appearance of sepsis, probably due to the development of concomitant severe leukopenia (white blood cell count less than 1,OOO/pl), necessitating cessation of the therapy as no effect on the platelet count was observed. On June 30, 1982, and, again on August 3, 1982, injection of 32P resulted in a reduced platelet count (8 X 105/pl) and a stable white blood cell count (3,500/~1), with improvement in the patient’s condition. However, at the time of this writing thrombocythemia has again developed (platelet count 1.7 to 2.6 X 106/~I). No evidence of the blastic phase has appeared.
Comment: This case of Ph’-positive chronic myelocytic leukemia has several interesting facets, from both a cytogenetic and clinical viewpoint. The appearance of two additional chromosomal changes,
CHROMOSOME
ANALYSIS
IN HEMATOLOGIC
DISORDERS--SANDBERG
ET AL
b
Figure 4. Karyotype (G banded) of a leukemic cell from Patient 3 with acute nonlymphocytic leukemia. The major karyotypic change is an 8;2 1 translocation (broken arrows) seen in the M-2 type of acute myeloblastic leukemia; the 9qanomaly (solid arrow) usually suggests a graver prognosis when the anomaly appears, as it did in this patient.
i 1 i j 1 / 1
14
”
15
lb
r
rJ * m
20
-
i.e., 9q- and 13q-, was not associated with the development of the blastic phase. However, concomitant with the development of the chromosome changes, thrombocythemia, which was not present during the initial four years of her disease, became a distressing part of the chronic myelocytic leukemia and has been difficult to control. It is possible that the thrombocythemia represents an unusual manifestation of a blastic phase, constituting in a way an increased proliferation of megakaryocytic elements, instead of the usual increase in myeloblasts and related cells seen in a bona fide blastic phase. Patient 3. This patient, a 52%year-oldman with acute nonlymphocytic leukemia, is of interest because his case points to the value of obtaining chromosome analysis in the follow-up of such patients. The patient’s history consisted of fatigue, weakness, and easy bruising for a period of about a month prior to examination. On January 15, 198 1, his white blood cell count was 45,4OO/~l with 29 percent blasts (containing Auer rods) in the blood, hemoglobin level 7.9 g/dl, hematocrit 25 percent, and platelet count 67,OOOl~l. The patient began to receive chemotherapy (daunomycin, cytosine arabinoside, and 6thioguanine); complete remission was achieved as evidenced by the normal blood picture on March 10, 1981, with a white blood cell count of 9,8OO/pl and no blast forms, a hemoglobin level of 22.3 g/dl, hematocrit of 34 percent, and platelet count of 2OO,OOO/@l.However, on April 20, 1981, when the patient was admitted for consolidation chemotherapy, the white blood cell count was 5,600/& the hematocrit 36 percent, and the platelet count 285,OOO/pl; the bone marrow contained 23 percent of cells with the abnormal karyotype. In July, the patient had an asymptomatic relapse of the disease. During the summer of 1981, the patient received 12 platelet transfusions because of severe thrombocytopenia,
-
but the platelet count never rose above 25 X lo”/pl. The white blood cell count during that period ranged from 1,5OO/pl to 6,0OO/~l. The patient died on August 27, 1981. When originally seen (January 16, 1981), the patient had a mixture of cells in the blood and bone marrow consisting of cytogenetically normal cells, as well as those having the (8;21)(q22;q22) translocation, not an uncommon finding in acute myeloblastic leukemia of the M-2 variety, but also being accompanied by deletion of the long arm of chromosome 9 (9q-) at band q22 (Figure 4). Generally, patients with the 8;21 translocation have a better prognosis than most other patients with acute nonlymphocytic leukemia [ 1,2], have more favorable responses to therapy and, hence, show and have maintenance of complete remission more readily than patients with other forms of acute nonlymphocytic leukemia. However, in the presence of other karyotypic changes, in this case 9q-, the outlook for these patients changes radically, for they tend to have rather short remissions and survival. In fact, this was the case in our patient, who began to receive chemotherapy in midJanuary 1981, had complete remission about mid-March of that year, but had a partial relapse a month later and an overt relapse in July 198 1. Cytogenetic analysis performed on marrow samples before July (on April 20, 1981 and May 28, 1981) indicated relapse of the leukemia by the appearance of a substantial number of cells with the chromosomal changes just described, i.e., 8;21 translocation and 9q- deletion; thus, the bone marrow contained 23 percent blast forms in April, 50 percent in May, and nearly 100 percent in July. On July 24, 1981, the marrow showed exclusively cells with the 8;21 translocation and 9q- deletion.
Comment: The 8;21 translocation has been shown to characterize a specific subgroup of patients with acute nonlymphocytic leukemia, i.e., acute myeloblastic leukemia of the M-2 type. These patients with acute myeloblastic leukemia have a relatively good prognosis
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CHROMOSOME
ANALYSIS
IN HEMATOLOGIC
DISORDERS-SANDSERG
ET AL
Flgure 5. Karyotype (G banded) of a leukemic blood cell from Patient 4 with malignant lymphoma. included in the abnormalities are a 74qi- (thick arrow}, an i(3q) (thln arrow), and a translocation between chromosomes 8 and 22 (curved arrow).
when compared with other patients with acute nonlymphocytic leukemia, readily have responses to therapy, and have long complete remissions, and usually have Auer bodies in the leukemic cells. However, when additional karyotypic changes appear, the clinical course usually worsens and the disease becomes difficult to control with chemotherapy. In Patient 3, the appearance of the 9q- anomaly was associated with progression of the disease. Patient 4. A 61-year-old Hispanic male gardener was admitted with a possible diagnosis of acute myeloblastic leukemia or related disorder, made on the basis of bone marrow examination. He was admitted to the Palo Alto Veterans Administration Hospital on June 12, 1982 from another hospital for an evaluation of “hypersplenism and chronic lymphocytic leukemia.” The patient had a history of malaise, lethargy, intermittent epistaxis, fever and chills, diaphoresis, and cough for three to four months, accompanied by a weight loss (12 to 15 pounds) due to decreased appetite over the preceding six months. When first seen, the patient had “massive splenomegaly and an abnormal complete blood count.” On admission to the Palo Alto Veterans Administration Hospital, the patient had no hepatomegaly but did have splenomegaly with the spleen edge at the iliac crest. Before admission, the white blood cell count was 14,500/@ with 11 percent blasts, 14 percent promyelocytes, 35 percent lymphocytes, 14 percent atypical cells, 32 percent segmented cells, 2 percent monocytes, 6 percent eosinophils, and IO percent “smudge” cells; the hemoglobin level was 9.0 g/dl, and the hematocrit was 27.6 percent. Bone marrow biopsy and aspiration specimens obtained on June 14, 1982 contained “multiple histiocytes among many abnormal-looking lymphocytes with indented and elongated nuclei.” The blood cells were terminal deoxyri-
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bonucleotidyl transferase-negative. The diagnosis of Lennett’s lymphoma was agreed upon. Treatment with cyclophosphamide, vincristine, procarbazine, and prednisone led to a greatly improved clinical state, although at the time the patient decided to go home, he still had a white blood cell count of 23,1OO/pl with 12 percent polymorphonuclear cells, 1 percent band forms, 78 percent lymphocytes, 3 percent monocytes, 2 percent eosinophils, and 4 percent promyelocytes, a hematocrit of 30 percent, and a platelet count of 84,OOOI~l. Karyotyping of blood cells examined on June 15, 1982 after incubation without phytohemagglutinin revealed the following: 46,XY/45,XY,-22,lp+,dup(lq)(q21+q23), i(3q),t(8;22)(pl1.2;q11.2),14q+ (Figure 5). The source of the extra material on the long arm of chromosome 14 could not be established with certainty. The results of the cytogenetic studies were obtained at a time when the exact diagnosis in this patient was uncertain. The results indicated that in all probability the patient did not have acute or another type of leukemia but probably some type of lymphoma, as based on the presence of the 14q+ anomaly, which is extremely rare in acute myeloblastic leukemia and other types of acute nonlymphocytic leukemia [ 71. Subsequent re-examination of the abnormal cells in the blood and a re-evaluation of the nature of the cells in the marrow revealed the most likely diagnosis to be malignant lymphoma, mixed cell type with high content of epithelial cells. Thus, examination of the bone marrow revealed it to be of normal cellularity. The myeloid-to-erythroid ratio was low [ 1,8]. The differentiation of the granulocytes and erythroblasts was normal. Sixty-two percent of the nucleated cells seemed to be abnormal lymphocytes (10 to 12 p in diameter) with basophilic cytoplasm, fine reticular chromatin, indented nuclei, and several nucleoli. These abnormal cells appeared to be compatible with those seen in lymphosarcoma cell leukemia or lymphoma. The disease has responded well to
CHROMOSOME
Figure 6. Blood and marrow leukemic cells of Patient 5. The monocytoid nature of the cells is evidenced by the folded nuclei and general appearance of the cells (Wri@t’s stain; original magnifi~tion X 1,200, reduced by 35 percent).
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therapy and is in complete remission at the time of this writing. Comment: The 14q-t anomaly has been shown to be the most common and consistent karyotypic change in various lymphomas, the incidence being particularly high in lymphomas of B cell origin, although 14q+ has also been seen in T cell lymphomas [ 1,7,8]. The origin of the extra material on chromosome 14 varies and the donor chromosome may ultimately be shown to have specificity, akin to that of chromosome 8 in Burkitt’s lymphoma and chromosome 18 in follicular lymphoma. The translocation between chromosomes 8 and 22 is rare in acute nonlymphocytic leukemia and is more likely to occur in lymphoma. In our patient, the short arm of chromosome 8 was involved in the translocation, whereas in most lymphomas with involvement of this chromosome, the long arm is characterized by a break at band q24. The other cytogenetic changes observed in our patient (lp+,duplq,i3q) may occur in both myeloid and lymphoproliferative disorders [l] and, thus, cannot be used as differential points in the diagnosis. In toto, the chromosomal changes observed in this patient are rather characteristic of lymphoma, in this case a lymphoma of mixed cell type. Patient 5. This patient, a 19-year-old man, has presented a rather interesting clinical condition, in that when he was admitted, the exact diagnosis was not readily ascertained. The patient was first seen on April 19, 1982, with a history of anorexia and left flank pain of very short duration. At that time, the blood findings showed a white blood cell count of 118,OOO/yl. The patient denied any other symptoms such as night sweats, fever, bone pain, or unusual bleeding. Past history was noncontributory.
On physical examination, the patient was a well-developed young man with a few ecchymoses. The liver was definitely enlarged (8 cm below the costal margin) and the tip of the spleen was palpable with a splenic rub being audible. Some adenopathy was also found. At the time of examination, the patient’s hemoglobin level was 14.9 g/dl, the hematocrit 41 percent, and the white blood cell count 157,3OO/~l with approximately 80 percent atypical cells. These cells were pleomorphic, with some appearing of lymphoid nature and others more myelomonocytic. The platelet count was 155,OOO/~l. During the patient’s hospitalization, the white blood cell count rose to nearly half a million in a week; bone marrow examination revealed relatively good erythropoiesis and presence of megakaryocytes. However, a large number of abnormal cells was present and these are thought to be myelomonoblastic in nature. Chemical stains of the peripheral blood suggested monocytic disease, as only testing for nonspecific esterase activity showed positive results. Periodic acid-Schiff, Sudan black, peroxidase, and specific esterase stains all demonstrated negative findings. Terminal deoxyribonucleotidyl transferase activity was very low. During the ensuing days, the spleen definitely increased in size, the hematocrit dropped to 34 percent, and the platelet count remained in the range of 150,000/~1. Treatment with daunomycin, cytosine arabinoside, and thioguanine was initiated, and within five days, his white blood cell count dropped to 22,9OO/pl. A reevaluation of the hematologic smears of the blood and marrow revealed about 70 percent of the cells in the blood to be mononuclear with slightly blue cytoplasm (Figure 6). The cells resembled to some extent those seen in S&ary syndrome or other forms of leukemic lymphoma or lymphosarcoma. A bone marrow specimen obtained on April 19, 1982 was shown to be markedly hypercellular with 30 percent of the nucleated cells belonging to the abnormal series described before. About 30 percent of the cells belonged to
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Figure 7. Karyotype (G banded) of a marrow cell from Patient 5 with acute myelomonocytic leukemia is shown at /eft, and the original chromosomal abnormality present in the leukemic cells of this patient is shown at right. The abnormality consisted of a (2;5)(p23;q35) translocation. At left are shown the karyotypic changes seen during a recent relapse (December 3, 7982}, i.e., the original 2;5 translocation (thin arrows) and a new translocation involving chromosomes 15 and 16 (thick arrows). In addition, partial trisomy of a chromosome 7 (left) consisting of duplication of 923-33 and + 7 are also present.
the erythroblastic series. The megakaryocytes appeared to be somewhat increased. The patient is now thought to definitely have had acute myelomonocytic leukemia with recent central nervous system involvement requiring the intrathecal administration of cytosine arabinoside. The latter led to a much improved status, with the maintenance therapy consisting of cytosine, vincristine, and prednisone. Throughout this period, the bone marrow has shown a remission picture. The patient had a relapse in early December 1982, including impressive lymphadenopathy and skin lesions. Since then, his condition has been in and out of remission, with each relapse being characterized by the aforementioned skin lesions and adenopathy. Considerable pleomorphism of the cells was observed in the blood during relapse, large cells being present in substantial number. Cytogenetic examination of the unstimulated (no phytohemagglutinin) blood cells in a sample obtained on April 20, 1982 revealed an abnormal karyotype consisting of a translocation between chromosomes 2 and 5, i.e., (2;5) (p23;q35) translocation (Figure 7). This finding appeared to be more compatible with those translocations observed in some forms of acute nonlymphocytic leukemia than in lymphoma. In particular, involvement of chromosome 5 occurs with a high frequency in acute nonlymphocytic leukemia [9, lo]. Furthermore, the translocation of the type observed in this patient, i.e., 2;5 translocation, and the lack of other chromosomal changes, combined to indicate that the most likely diagnosis in this patient was an acute leukemia of soma form, probably of the monocytic variety. Blood cells stimulated with phytohemagglutinin had a normal male karyotype.
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At the beginning of December 1982, the karyotypic findings in this patient progressed and underwent a radical change. The pseudodiploid cells seen previously, although still present, constituted a minority of the metaphases, with cells containing 47 (33 percent) and 78 (30 percent) chromosomes predominating. The cells with 47 chromosomes, in addition to the 2;5 translocation, developed a (15;16)(q22;p13.3) translocation (Figure 8) whereas the cells with 78 chromosomes, although still containing the 2;5 translocation, did not have the 15;16 translocation and showed only numeric changes consisting of extra chromosomes in every autosomal group, an extra X and a missing Y. The hyperploid cells with 78 chromosomes contained four normal number 15 and three normal number 16 chromosomes. Thus, it appears that in this patient, two divergent karyotypic progressions occurred, one with the 15; 16 translocation and the other through numeric changes only; however, the leukemic origin of the cells is evidenced by the presence of the 2;5 translocation, seen in the originally examined material, in both new clones. It is possible that extramedullary relapse of the acute myelomonocytic leukemia occurred in this patient (in lymph nodes, skin) and that the hyper-triploid clone with 78 chromosomes originated thusly and accounted for the pleomorphic blood cell picture. Comment: Acute myelomonocytic leukemia is a somewhat complicated entity in that this group of leukemias may be rather heterogeneous in character; thus, it is not surprising that the cytogenetic findings have not revealed any specificity akin to that in acute myeloblastic leukemia, acute monoblastic leukemia, and
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1
&
7
a
6s Figure 8. Katyotype (G banded) of a leukemic cell of Patient 6, containing a number of complicated chromosomal changes (see Figure 9). Arrows point to the three abnormal chromosomes whose genesis is shown schematically in Figure 9.
4: ..
.Q”
19
20
I
-------.~
(p14;ql l),t(7;16)(p14;pl3.3) (Figure 8). Thus, the short arm of chromosome 12 was lost and most of the long arm of chromosome 12 was translocated to an abbreviated chromosome 7. Part of the short arm of chromosome 7 was translocated to the short arm of chromosome 16 and another part to the short arm of chromosome 19 (Figure 9). The
acute promyelocytic leukemia [ 1,2,11]. In some patients, involvement of the long arm of chromosome 11 (1 lq-) has been described and in these the monocytoid element is rather clear and evident [ 121. Monosomy of chromosome 7 is frequent in acute myelomonocytic leukemia [ 1,9, lo] and may be indicative that such cases are possibly secondary in nature, since -7 has been shown to be a reflection of a leukemia secondary to drugs or toxic agents [9, lo]. The chromosomal findings in our patient, i.e., 2;5 translocation, are of interest, for they have not been described in acute myelomonocytic leukemia, although involvement of chromosome 5 is not rare in acute nonlymphocytic leukemia, particularly in leukemias complicating other diseases. Both 5qand -5 anomalies are seen often in secondary leukemias due to either radiation or chemotherapy [ 9, lo].
complexity of the chrornosotnal changes is rather unique and pointed to a definite leukemic process in operation. Comment:
The involvement
of chromosomes
5, 7,
Pallent 6. A 66-year-old man, a retired railroad boxcar cleaner (probably exposed to carbon tetrachloride), was first seen for severe pancytopenia and thought to have a preleukemic or leukemic condition. At the time of diagnosis, the bone marrow was found to be hypoplastic for erythroid, myeloid, and megakaryocytic elements, with mature lymphocytes, prolymphocytes, and a few lymphoblasts being seen in the marrow. The platelet count at that time was 45,OOO/~l, the white blood cell count 1,OOo/l.~lwith IO percent segmented cells, the hemoglobin level 5.5 g/dl, and the hematocrit 18 percent. The chromosomal findings determined on September 14, 198 1 were complex and quite interesting. The marrow contained a mixture of normal and abnormal cells, with the most likely description of the abnormal karyotype being 45,XY,-l2,del(l2)(qter~qll),t(7;19)(p21;p13.5),t(7;12)
12
1
16
19
7gure 9. Schematic presentation of the karyotypic changes seen in the leukemic cells of Patient 6 shown in Figure 8. As shown, the short arm of one chromosome 12 was lost, with the long arm being translocated to an abbreviated short arm of a chromosome 7. The latter had lost most of its short arm through translocations to chromosomes 16 and 19, with the part to chromosome 16 being inverted.
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Figure 10. Karyotype (G banded) of a marrow cell of Patient 7 with a myeloproliferative disorder (? preleukemia). The abnormalities consist of two abnormal (1p-l) number 1 chromosomes (arrows).
and 12 in Patients 5 and 6 may be indicative that the hematologic processes in these two cases, i.e., acute leukemias, may be related to some toxic exposure, since the involvement of these chromosomes has been shown to be rather frequent in these conditions [%lOl. The most likely interpretation of the chromosomal findings in Patient 6 is that they resulted from a toxic agent (perhaps carbon tetrachloride), which led to the bone marrow hypoplasia and what is probably a background of a smoldering secondary leukemia. Patient 7. A 77-year-old man presented with a complicated clinical picture of either preleukemia or an unusual myeloproliferative disorder, the latter shown subsequently to be myelofibrosis. Bone marrow biopsy performed on June 25, 1982 revealed alternating areas of hypercellularity and increased fibrosis. Treatment with allopurinol was begun. The karyotype was shown to be 46,XY/46,XY,lp+ (6 percent of
the cells) and 46,XY,lp+,lp+ (85 percent of the cells) (Figure 10). These karyotypic findings pointed to a definitely malignant hematologic disease, probably in the myeloid series, primarily based on the frequent involvement of chromosome 1 in such patients.
At the time of the chromosome analysis, the patient’s white blood cell count was 16.6 X 103, the marrow very fibrotic, and the clinical picture thought not to be that of a preleukemia. The patient has been uncooperative both regarding any further diagnostic procedures or regarding follow-up. As of the date of this writing, the patient’s condition has remained pretty much the same, with a hemoglobin level of 10 g/dl, a white blood cell count of 17,OOO/~l without blasts in the blood, and moderate hepatosplenomegaly.
Comment: Involvement of chromosome 1 is one of the most frequent in hematologic disorders [ 1,131, including trisomy (i- 1) of the chromosome and morpho-
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logic changes of either the short or long arm, but much more often of the long arm. Our Patient 7 is of further interest because some of the cells contained an extra copy of the 1p-t chromosome. Thus, in this patient, the chromosome changes pointed to a malignant hematologic disorder in the general category of a dysmyelopoietic or myeloproliferative syndrome, but its exact nature remains uncertain. COMMENTS This study demonstrated several “practical” points, among which is the feasibility of sending blood and marrow samples for cytogenetic analysis from considerable distances (e.g., Oakland and Palo Alto, California). The success rate in obtaining optimal cytogenetic information with such samples has been high. A crucial factor is to determine the express system of transportation that works expeditiously and reliably for a particular location; it is important to “educate” the personnel involved in transporting and delivering the samples about such crucial facets as the necessity for speed, the need to avoid freezing the samples (or overheating them), and the aim of having the samples in the cytogenetic laboratory within 24 hours or less. With proper attention and interest on the part of all involved in obtaining and shipping of the blood or marrow samples, this system can and has been made to work in Phoenix and Buffalo. There is no reason why a similar system cannot be established in other centers in the United States or other countries. Other points raised and demonstrated in this report are those associated with each of the seven representative cases of hematologic disorders in which the
CHROMOSOME ANALYSIS IN HEMATOLOGICDISORDERS-SANDBERG ET AL
in bone marrow and/or blood samples of patients who live considerable distances from a cytogenetic laboratory. Certainly, the information gained is more than worth the cost and effort. Even though establishment of the presence or absence of a Ph’ chromosome in chronic myelocytic leukemia has become a “routine” procedure in hematology and, in fact, failure to perform this analysis may constitute a form of malpractice in some locations, clinicians have not taken advantage of a similar approach in other hematologic disorders, particularly the acute leukemias. The practical application of karyotypic findings in the diagnostic and prognostic aspects of the acute leukemias has been spelled out in several international workshops on the chromosome changes in these diseases [2,3,5]. Yet, only a minority of oncologists and hematologists have taken advantage of the cytogenetic information in the acute leukemias and related disorders. There are a number of reasons for this situation, as compared with the determination of the Ph’ chromosome in chronic myelocytic leukemia, which possibly account for the infrequency of performing chromosome analysis in acute leukemia and similar disorders. These include the more technically difficult side of examining the chromosomes in acute leukemia cells versus those of chronic myelocytic leukemia, relatively complex karyotypic changes in acute leukemia, as well as their more complex interpretation, and lack of appreciation of the meaning and nature of the cytogenetic abnormalities in the acute leukemias. Hopefully, the results presented herein will convince clinicians to avail themselves of what we think is a critical and crucial aspect of leukemia management.
cytogenetic findings were useful diagnostically and therapeutically. Briefly, the following patients were presented: Patient 1: chronic myelocytic leukemia with a Phi chromosome and two additional translocations, 16; 18 and X;lO, heralding an imminent blastic phase; Patient 2: chronic myelocytic leukemia with a Ph’ chromosome and two marker chromosomes (9q- and 13q-) associated not with the blastic phase but with thromobocythemia; Patient 3: acute myeloid leukemia of the M-2 type with an 8;21 translocation and then a 9q- marker indicating imminent relapse; Patient 4: malignancy of uncertain type with a 14q+ marker chromosome indicative, not of acute myeloblastic leukemia, but of lymphoma; Patient 5: leukemia of uncertain type with a 2;5 translocation consistent with acute nonlymphocytic leukemia, specifically acute myelomonocytic leukemia; Patient 6: pancytopenia probably due to exposure to organic solvents, with 12q- marker and three translocations (7; 12, 7; 16, and 7; 19) indicative of smoldering secondary leukemia; and Patient 7: myelofibrosis, with a lpi- marker associated with a dysmyelopoietic disease. In each of the seven cases presented, the exact diagnosis was not readily apparent when the patients were first seen or when a change in the clinical condition occurred. In several patients, confusion due to the complicated histologic features, cinical condition, or signs prevented the pathologist and oncologist from arriving at a specific diagnosis with confidence. Examination of the chromosomes was of considerable help, i.e., ruling out a diagnosis being considered in several cases, pointing to the type of disease present in others, and establishing the diagnosis with specificity in some. Thus, the utilization of cytogenetic data, when considered in conjunction with the clinical, pathologic, and laboratory data regarding a particular patient, can be of considerable value in the management of patients with various blood disorders. It is hoped that cytogenetic studies of patients with hematologic disorders will find an ever-widening application, particularly, as already indicated, since such studies can be readily performed
ACKNOWLEDGMENT This study would not have been possible without the collaboration of the following clinicians: Teresita C. Barnett, Sun City, Arizona; Robert D. Ligorsky, Phoenix, Arizona; Jerry A. Olshan, Phoenix, Arizona; Donald E. Rediker, Palo Alto, California; Philip P. Scheerer, Phoenix, Arizona.
REFERENCES 1. 2.
3.
4.
Sandberg AA: The chromosomes in human cancer and leukemia. New York: Elsevier North-Holland, 1980. Second International Workshop on Chromosomes in Leukemia (1979): Cytogenetic, morphologic, and clinical correlations in acute non-lymphocytic leukemia with t(8q--:21q+). Cancer Genet Cytogenet 1980; 2: 99-102. Third International Workshop on Chromosomes in Leukemia (1980): Chromosomal abnormalities in acute lymphoblastic leukemia: structural and numerical changes in 234 cases. Cancer Genet Cytogenet 1981; 4: 101-110. Sandberg AA, Abe S: Cytogenetic techniques in hematology. Clin Haematol 1980; 9: 19-38.
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First International Workshop on Chromosomes in Leukemia, Helsinki, Finland (1977): Cancer Res 1978; 38: 867868. Sandberg AA: The X chromosome in human neoplasia. ineluding sex chromatin and congenital conditions with X chromosome anomalies. In: Sandberg AA, ed. The cytogenetics of the mammalian X chromosome. New York: Alan R. Liss, 1983; 459-498. Mitelman F, Levan G: Clustering of aberrations to specific chromosomes in human neoplasms. IV. A survey of 1,871 cases. Hereditas 1981; 95: 79-139. Yunis JJ, Oken MM, Kaplan ME, Ensrud KM, Howe RR,
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Theologides A: Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkin’s lymphoma. N Engl J Med 1982; 307: 1231-1236. Rowley JD, Golomb HM, Vardiman JW: Nonrandom chromosome abnormatiii in acute l&emia and dysmyelopoietic syndromes in patients with previously treated malignant disease. Blood 1981; 58: 759-767. Sandberg AA, Abe S, Kowalczyk JR, Zedgenidze A, Takeuchi J, Kakati S: Chromosomes and causation of human cancer and leukemia. L. Cytogenetics of leukemias complicating
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11.
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other diseases. Cancer Genet Cytogenet 1982; 7: 95136. Hagemeijer A, H&hlen K, Sizoo W, Abels J: Transkxation (9; 1 l)(p21;q23) in three cases of acute monoblastic leukemia. Cancer Genet Cytogenet 1982; 5: 95-105. Berger R, Bemheim A, Weh HJ, Daniel MT, Flandrin G: Cytogenetic studies on acute monocytic leukemia. Leuk Res 1980; 4: 119-127. Rowley JD: Abnormalities of chromosome no. 1 in haematological malignancies. Lancet 1978; I: 554-555.