Clonal Changes in Chronic Granulocytic Leukemia in Blastic Transformation and During Remission

GERMAN BELTRAN, MD,' AND MARIA VARELA, MDt

Cytogenetic studies of the peripheral blood and marrow of a patient with chronic granulocytic leukemia in blastic transformation revealed the following abnormalities: 1) a Philadelphia chromosome with the usual 9:22 translocation; 2) a three break rearrangement (insertion) of chromosome #11; and 3) a deletion of the short arm in chromosome #16. Clinical and hematologic remission developed after three courses of intermittent chemotherapy with transient but complete bone marrow aplasia. Studies performed during remission revealed two cellular clones. One had the same karyotype already described and the second showed only a Ph' chromosome. We postulate that the pretreatment clone evolved from Ph'- positive cells in this patient with chronic granulocytic leukemia during a short, undiagnosed stable phase. Treatment allowed the reappearance of the Ph'-positive clone. The second abnormal clone was apparently partially eradicated but normal cells did not repopulate the bone marrow, suggesting that either the Ph'-positive cells were greatly resistant to therapy or that there were no remaining normal myelogenous stem cells. In addition, we found that there have been few reports of abnormalities involving chromosomes #I1 and #16, and that an inserting chromosome #I1 has never been reported.

Cancer 46:1590-1593, 1980.

N THIS REPORT, we describe a case of a patient with I chronic granulocytic leukemia who presented in blastic transformation. Cytogenetic studies revealed the presence of a Ph' chromosome, a structurally abnormal chromosome # 1 1, plus a deletion of chromo- some #16. These abnormalities persisted in some cells after the patient achieved a clinical and hematologic remission of her blastic transformation.

Case Report

A 50-year-old black woman presented to her physician with pallor of the skin and mucous membranes and an enlarged spleen, the tip of which was felt 8 cm below the left costal margin. Laboratory studies revealed anemia and marked leu- kocytosis. The patient was transferred to the Tulane Medical Center Hospital in October 1977. Initial laboratory results included: 9.7 g/dl; reticulocyte count, 0.2%; leukocyte count, 340,000/mm3 with 19% blasts, 16% promyelocytes, 1%

From the Section of Hematology-Oncology, Department of Medi- cine, and the Hayward Genetics Center, Department of Pathology, Tulane University School of Medicine, New Orleans, Louisiana.

Supported in part by the Perkins Leukemia Research Fund, USPHS Grant No. CA03389, and the Hayward Foundation.

* Professor of Medicine. i Associate Professor of Pathology. Address for reprints: German Beltran, MD, Section of Hematol-

ogy-Oncology , Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana 701 12.

The authors thank Mrs. Delma Herrera and Mrs. Viola Blackman for their technical assistance.

Accepted for publication November 7, 1979.

myelocytes, 2% metamyelocytes, 33% neutrophiles, 4% bands, 2% eosinophiles, 18% basophiles, 3% monocytes, and 2% lymphocytes; and platelet count, 175,000/mm3. Bone marrow aspirated from the sternum showed hypercellularity, abundant megakaryocytes, and a differential count of 15% myeloblasts, 13% progranulocytes, 20% myelocytes, 6% metamyelocytes, 8% bands, 1 1% neutrophiles, 2% eosino- philes, 15% basophiles, 5% monocytes and monocytoid cells, 3% lymphocytes, and 2% erythroid precursors. The leuko- cyte alkaline phosphatase score on the peripheral blood film was 7 (range, 0-400). Results of cytogenetic studies are reported below. A diagnosis of chronic granulocytic leukemia in blastic transformation was made and chemotherapy was begun with weekly doses of vincristine and daily doses of prednisone, hydroxyurea, and cytosine arabinoside. Therapy continued until complete marrow aplasia developed. When the marrow recovered with decreased but persistent blastic infiltration, two additional courses of therapy were given. By treatment day 63, the spleen could no longer be felt, the hemo- globin was 8.3 g/dl; reticulocyte count, 0.1%; leukocyte count, 15,600/mm3, with 88% neutrophiles, 7% lymphocytes, 2% basophiles, and 3% eosinophiles; and platelet count, 160,000/mm3. Bone marrow aspirated from the sternum showed normal megakaryocytes, 4% blasts, 9% progranulo- cytes, 26% myelocytes, 9% metamyelocytes, 4% bands, 11% neutrophiles, 6% eosinophiles, 8% basophiles, 1% lympho- cytes, 2% monocytes, and 20% erythroid precursors. The patient was considered to be in remission from the blastic transformation. The anemia was ascribed to residual myelo- suppression and the leukocytosis to reversion to the chronic phase of chronic granulocytic leukemia.

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We attempted intermittent chemotherapy for maintenance with the combination of cytosine arabinoside and prednisone given for five days, and vincristine given once on the first day of each course. Vincristine could not be given after the first course due to a moderately severe neurotoxic reaction and prednisone was discontinued after the sixth course because of moderately severe neuropsychiatric manifestations and symptomatic progressive osteoporosis. Courses were given at the shortest feasible intervals, usually 17-26 days. After each course of treatment, there was a mild decrease in the leukocyte count but a rapidly increasing granulocyte count promptly followed. Platelet counts remained normal or moderately increased and mild anemia persisted.

Although there were moderately severe complications from therapy, the patient remained free from infection, hemor- rhaging, incapacitating anemia, or organomegaly. Cytoge- netic studies of bone marrow and peripheral blood were repeated during remission in May and September 1978. Shortly after the last study in September 1978, immaturity of the leukocytes and progressive thrombocytopenia and anemia developed. Bone marrow examination performed in October 1978 showed an infiltration with 22% myeloblasts and 25% progranulocytes. The patient was considered to be in blastic transformation again. Two courses of adriamycin and cyto- sine arabinoside and two courses of adriamycin and iphos- phamide failed to induce another remission and the patient died in January 1979.

Methods

Chromosome analyses were performed according to standard techniques. Peripheral blood cultures were incubated at 37°C for 72 hours while bone marrow specimens were incubated for 24 hours, both i n the absence of phytohemagglutinin. Each slide was banded with trypsin-Giemsa using a modification of the Sea- bright technique.'" A minimum of 30 metaphases were analyzed in each study. Five to seven karyotypes of each cell line were prepared from the best banded cells.

Results

Cytogenetic studies were undertaken on three differ- ent occasions. The first chromosomal study was done on peripheral blood leukocytes shortly after the patient was first seen. At this time, all the cells analyzed were Phl-positive with the usual 9;22 translocation. Two additional and consistent structural abnormalities, one involving chromosome #11 and the other, chromosome # 16, were present. The structurally abnormal chromo- some #I1 was interpreted as resulting from a three break rearrangement. One break occurred in the long arm at 1 lq23 and the others in the short arm at 1 lp15 and llp12. The segment between llp12 and l lp15 was inserted at band 1 lq23. A deletion involving most of the short arm was present in chromosome #16. The karyo-

type signature was 46,XX,t(9;22)(q34;ql l),ins( 11)- (q23p15p12),del( 16),(pl 1)5 (Fig. 1).

Chromosome analysis of bone marrow performed in May 1978 revealed 16 cells with the same karyotype a5

seen previously. Fourteen metaphases were normal except for the presence of the Ph' chromosome-46,- XX,t(9;22)(q34;qll) (Fig. 2).

The third chromosome study, of both peripheral blood leukocytes and bone marrow, was done in September 1978. The results were similar to those of the previous studies. In 7.5% of the 50 blood cells and in 70% of the 40 marrow cells analyzed, the only abnormal- ity was the presence of the Ph' chromosome. The other 25% of the blood cells and 30% of the marrow cells showed a loss of most of the short arm of chromosome #16, the abnormal #11, plus the Ph' chromosome.

Discussion

Occasionally, a patient with chronic granulocytic leukemia will have a very short and abortive stable phase and the disease is diagnosed after a blastic crisis has already developed.6 Some clinical and hematologic characteristics which appear to distinguish this type of case from that of de novo acute leukemia include signif- icant splenomegaly, absence of severe thrombocyto- penia, some degree of differentiation of leukocytes in both bone marrow and the peripheral blood, marked elevation of the leukocyte count, and basophilia. These characteristics were found in our patient.

Cytogenetic abnormalities in addition to the presence of the Ph' chromosome have been detected in patients with chronic granulocytic leukemia in blastic trans- formation4 and have infrequently involved chromo- somes # 11 and # 16. A trisomy for chromosome # 1 1 was found in 4 of 67 cases reviewed by Mitelman et ( 1 1 , ~ In the same series, only 1 instance of a trisomy for chromosome #16 and 1 case of loss of chromosome #16 were seen. There was no abnormalities involving chromosome #11 or #16 among 1.5 cases reported by Prigonina.' In a review of 93 cases, RowleyH found 3 with a trisomy, 3 with rearrangements involving chro- mosome #1 I , and 2 with a trisomy for chromosome #16. A rearrangement of chromosome # I 1 similar to that described for our patient was not reported in any of these series.

Treatment in the present case resulted in marked clinical and hematologic improvement and eventually in a remission of the blastic transformation. In view of the relative instability of the disease suggested by the unusually rapid increase in granulocytes, a regimen of frequent courses of maintenance chemotherapy was instituted. The finding on repeated cytogenetic studies of two cell clones, one identical to that seen prior to

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treatment and the second showing only the Ph' chromo- some, was consistent with the following interpreta- tions. The development of a Phl-positive clone was promptly followed by the evolution of a second abnormal clone characterized cytogenetically by two additional abnormalities, i.e., the insertion involving chromosome # 1 1 and the short arm deletion of chromo- some #16. This second clone probably had a growth advantage and was associated with the rapid develop- ment of the blastic transformation. With treatment, the more malignant clone was partially eradicated, while the postulated initial 46,XX,Ph1 clone reappeared.

Clinical and hematologic remissions of the blastic crisis of chronic granulocytic leukemia seldom occur, and thus there is only limited information regarding the fate during remission, of the cellular clones found dur- ing the acute state of the disease. Sandberg et ~ 1 . ~ have suggested that they may become a permanent feature and that they may be epiphenomena of the blastic crisis rather than causally related to it. Such views were sug- gested by occasional reports of their persistence after

FIG. 1. Peripheral blood leukocyte karyotype (Tripsin-Giemsa banding). Note the rearranged chromosome 11 (arrow), the short arm dele- tion of chromosome 16 (arrow), and the Ph' chromosome (arrow).

remission.',2 Hagemeijer et ~ 1 . ~ have reported a case of cells exhibiting several cytogenetic abnormalities (49,- XY,+9 ,+ lo,+ 12) during blastic crisis which disap- peared after a remission and reappeared during relapse. This sequence of events in this case and in ours is consistent with the theory that therapy sup- pressed more malignant clones and allowed the return of the clinical and hematologic picture of chronic granu- locytic leukemia. However, the suppression of the vari- ant clones was only partial. In patients achieving remis- sion of blastic crisis, the cytogenetic picture might reveal a mosaic such as in our patient, or as in some of the previously reported cases, either a complete disap- pearance of the variant clones or their absolute persist- ence. The reasons for this differing behavior are not known but may represent varying degrees of sensitivity of the cellular clones to therapy. Whether there is a correlation between the cytogenetic picture during remission and the duration of the remission awaits fur- ther serial studies. In our patient, the rapid rise of the leukocyte count following each course of maintenance

No. 7 GRANULOCYTIC LEUKEMIA . Beltran and Varela

FIG. 2. Trypsin-Giemsa banded karyotype representative of the second clone of cells found in this patient. Note the presence of the Ph' chromosome and absence of all other abnormalities.

chemotherapy might have resulted from the failure to completely eliminate the seemingly more malignant cellular clone. Finally, the persistence of the Ph'-posi- tive clone, despite the aggressive chemotherapy for remission induction which resulted in three episodes of marrow aplasia, is in agreement with the previously observed difficulty in eradicating Phl-positive cells in patients with chronic granulocytic 1eukemia.l'

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